The Ras family regulates a wide variety of cellular functions that include cell growth, differentiation, and apoptosis. In this study, we identified a novel human gene named RAP2C, isolated from human testis cDNA library, and mapped to Xq26.2 by searching the UCSC genomic database. The RAP2C cDNA contains an open reading frame of 552 bp, encoding a putative protein of 183 amino acid residues. The predicted protein contains a RAS domain. By RT-PCR analysis in various tissues, RAP2C was found to be principally expressed in the liver, skeletal muscle, prostate, uterus, rectum, stomach, and bladder and to a less extent in brain, kidney, pancreas, and bone marrow. RAP2C protein was located in cytoplasm when overexpressed in COS-7 cells. Reporter gene assays showed that overexpression of RAP2C in HEK293T cells activated the transcriptional activities of serum response element (SRE). These results indicate that RAP2C is a novel member of the Ras family, belonging to the Rap branch of small GTPase proteins and may be involved in SRE-mediated gene transcription.

Lysophosphatidic acid acyltransferase (LPAAT) is an intrinsic membrane protein that catalyzes the synthesis of phosphatidic acid (PA) from lysophosphatidic acid (LPA). It is well known that LPAAT is involved in lipid biosynthesis, while its role in tumour progression has been of emerging interest in the last few years. To date, seven members of the LPAAT gene family have been found in human. Here we report a novel LPAAT member, designated as LPAAT-theta, which was 2728 base pairs in length and contained an open reading frame (ORF) encoding 434 amino acids. The LPAAT-theta gene consisted of 12 exons and 11 introns, and mapped to chromosome 4q21.23. LPAAT-theta was ubiquitously expressed in 18 human tissues by RT-PCR analysis. Subcellular localization of LPAAT-theta-EGFP fusion protein revealed that LPAAT-theta was distributed primarily in the endoplasmic reticulum (ER) of COS-7 cells. Furthermore, we found that the overexpression of LPAAT-theta can induce mTOR-dependent p70S6K phosphorylation on Thr389 and 4EBP1 phosphorylation on Ser65 in HEK293T cells.

The Ras family of small GTPases regulates a wide variety of cellular functions that include cell growth, differentiation, and transformation. In this study, we identified and characterized a novel member of Ras family named RHEBL1, belonging to the Rheb branch of small GTPase proteins. The cDNA sequence contains an open reading frame of 551 bp, encoding a putative protein of 183 amino acid residues. The expression pattern of RHEBL1 showed that it was ubiquitously expressed in 17 tissues. RHEBL1 gene encodes a 20.69 kDa protein, localized in cytoplasm when overexpressed in COS7 cells. Reporter gene assays showed that overexpression of RHEBL1 in HEK 293T cells strongly activated the transcriptional activities of NF-kappa B, while the mutant (D60K) only weakly activates NF-kappa B-mediated transcription. Our findings suggest that RHEBL1 is a positive regulator of NF-kappa B-mediated gene transcription.

We report here the cloning and characterization of the human gene DERP6, isolated from human testis cDNA library, and mapped to 17p13.1 by searching the UCSC genomic database. The DERP6 cDNA consists of 1486 nucleotides and has a 316-amino acids open reading frame. The Northern hybridization analysis showed that DERP6 was ubiquitously expressed in all the 16 adult tissues, especially highly expressed in heart, brain, liver, skeletal muscle and testis. DERP6 protein was located in cytoplasm when overexpressed in cultured cells. Reporter gene assays showed that overexpression of DERP6 in cells activated the transcriptional activities of p53. These results indicate that the human gene DERP6 may be involved in p53-mediated gene transcription.

In response to DNA damage, many DNA damage factors, such as MDC1 and 53BP1, redistribute to sites of DNA damage. The mechanism governing the turnover of these factors at DNA damage sites, however, remains enigmatic. Here, we show that MDC1 is sumoylated following DNA damage, and the sumoylation of MDC1 at Lys1840 is required for MDC1 degradation and removal of MDC1 and 53BP1 from sites of DNA damage. Sumoylated MDC1 is recognized and ubiquitinated by the SUMO‐targeted E3 ubiquitin ligase RNF4. Mutation of the MDC1 Lys 1840 (K1840R) results in impaired CtIP, replication protein A, and Rad51 accumulation at sites of DNA damage and defective homologous recombination (HR). The HR defect caused by MDC1K1840R mutation could be rescued by 53BP1 downregulation. These results reveal the intricate dynamics governing the assembly and disassembly of DNA damage factors at sites of DNA damage for prompt response to DNA damage.

Histone H3 lysine 56 acetylation (H3K56Ac) has recently been identified and shown to be important for genomic stability in yeast. However, whether or not H3K56 acetylation occurs in mammals is not clear. Here, we report that H3K56Ac exists in mammals. Mammalian H3K56Ac requires the histone chaperone Asf1 and occurs mainly at the S phase in unstressed cells. Moreover, SIRT1, which is a mammalian member of sirtuin family of NAD+-dependent deacetylases, regulates the deacetylation of H3K56. We further showed that proper H3K56 acetylation is critical for genomic stability and DNA damage response. These results establish the existence and functional significance of H3K56Ac in mammals and identify two regulators of this modification.

Topoisomerase II (Topo II) is required to separate intertwined sister chromatids before chromosome segregation can occur in mitosis1. However, it remains to be resolved whether Topo II has any role in checkpoint control. Here we report that when phosphorylated, Ser 1524 of Topo IIα acts as a binding site for the BRCT domain of MDC1 (mediator of DNA damage checkpoint protein-1), thereby recruiting MDC1 to chromatin. Although Topo IIα–MDC1 interaction is not required for checkpoint activation induced by DNA damage, it is required for activation of the decatenation checkpoint. Mutation of Ser 1524 results in a defective decatenation checkpoint. These results reveal an important role of Topo II in checkpoint activation and in the maintenance of genomic stability.

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1's role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc's transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression.

Mediator of DNA damage checkpoint 1 (MDC1) plays an important role in the DNA damage response (DDR). MDC1 functions as a mediator protein and binds multiple proteins involved in different aspects of the DDR. However, little is know about the organization of MDC1 complexes. Here we show that ataxia telangiectasia, mutated (ATM) phosphorylates MDC1 at Thr-98 following DNA damage, which promotes its oligomerization. Oligomerization of MDC1 is important for the accumulation of MDC1 complex at the sites of DNA damage. Mutation of Thr-98 (T98A) would abolish its oligomerization and result in a defect in DNA damage checkpoint activation and increased sensitivity to irradiation. Taken together, these results suggest that the oligomerization of MDC1 plays an important role in DDR and help understand the formation of proteins complexes at the sites of DNA damage.

SIRT1 regulates a variety of cellular functions, including cellular stress responses and energy metabolism. SIRT1 activity is negatively regulated by DBC1 (Deleted in Breast Cancer 1) through direct binding. However, how the DBC1–SIRT1 interaction is regulated remains unclear. We found that the DBC1–SIRT1 interaction increases following DNA damage and oxidative stress. The stress-induced DBC1–SIRT1 interaction requires the ATM-dependent phosphorylation of DBC1 at Thr 454, which creates a second binding site for SIRT1. Finally, we showed that the stress-induced DBC1–SIRT1 interaction is important for cell fate determination following genotoxic stress. These results revealed a novel mechanism of SIRT1 regulation during genotoxic stress.